The goal of this project is to understand the roles of RNA binding proteins in the post-transcriptional regulation of gene expression, through an analysis of the in vivo functions of proteins that contain RNA recognition motif (RRM) domains. RRM domains have been identified in many proteins involved in post-transcriptional regulation, and can mediate sequence-specific binding to RNA targets. We use genetics, biochemistry and molecular biology to study six Drosophila RRM proteins. Three of these proteins, HRB87F, HRB98DE and CAZ, are homologs of vertebrate proteins that bind nuclear pre-mRNA (hnRNP proteins). We have isolated mutations in both Hrb genes. The mutation in the Hrb87F gene completely deletes the coding region. Homozygous mutant flies are viable and fertile, demonstrating that the HRB87F protein is not essential. However, in the appropriate genetic background, these flies are sensitive to the dosage of HRB98DE. Using this observation, we performed a genetic screen and isolated a mutation in Hrb98DE. This mutation abolishes the expression of the protein, as shown by Western blots of protein extracts from adults. Homozygous or hemizygous individuals show somewhat reduced viability but, like HRB87F, HRB98DE is not essential. However, homozygosity for both mutations is lethal, demonstrating that flies require a minimal level of HRB proteins for survival and suggesting that the two HRB proteins are functionally redundant. In vitro studies have suggested that hnRNP A1, a vertebrate homolog of the HRBs, may be a general splicing regulator. Data from overexpression and reduced expression fails to support such a role for the HRB proteins. However, preliminary data suggest that in the appropriate sensitized genetic backgrounds, reduced Hrb gene dosage can modulate the expression of the Ultrabithorax gene, which regulates segment identity. The CAZ protein, the Drosophila homolog of hnRNP P2, shows widespread but not ubiquitous expression during development. We have identified a novel RRM-containing protein that interacts with the CAZ protein in a yeast two-hybrid selection screen, and that may be a specific partner in ribonucleoprotein complexes. We have also generated deletion mutants and transgenic fly strains for use in mutagenesis screens for caz mutants.